The dynamics of PSD components during synaptic activity were studied by pre-embedding Nanogold labeling of dissociated hippocampal neurons in culture. Depolarization with high K+ is typically used to activate neuronal cultures, but other protocols closer to physiological stimulation, such as application of NMDA and synaptic activation with glycine in the absence of Mg2+, are also used. Now that antibodies for nearly all major components of the PSD have been obtained and tested for compatibility for immunoelectron microscopy, a general picture of activity-induced reorganization of the PSD at the molecular level begins to emerge. Scaffold proteins such as PSD-95 and GKAP appear to be relatively stable during short-term activity, and constitute a layer 30 nm thick lying next to the post synaptic membrane where receptors are stabilized by associations with the scaffold. In contrast, other proteins deeper in the PSD change location during short-term activity. For instance, Shank moves closer and into to the PSD after stimulation. A study of the distribution of the scaffold protein, Homer, has been completed. Its long isoform is ubiquitous in the PSDs of spine synapses, but in a layer just deep to the central core of PSD-95 and receptors that many proteins move into during activity. Homer has many binding partners among PSD proteins, and its abundance puts it in a position to be a central scaffolding protein deeper in the PSD, just like the role of PSD-95 in the central core. This idea is supported by our finding that, unlike other PSDs proteins, Homer shows little movement in or out of its layer in the PSD upon acute stimulation. We had previously shown that while CaMKII translocates to the PSD during activity, SynGAP, another major regulatory, moves away from the PSD core. The changes induced by stimulation coincide with a marked increase of AMPA receptor labeling at the PSD, compatible with enzymatic as well as structural roles of CaMKII and SynGAP in regulating AMPA receptor trafficking at the PSD. Previous work on SynGAP was expanded, showing that two isoforms that differ in their binding to PSD-95 are both localized at the PSD core under basal conditions. Application of NMDA promotes their movement away from the PSD core, and this effect is blocked when CaMKII activity is inhibited, indicating mediation by CaMKII. Because the two isoforms of SynGAP are known to have opposing effects on synaptic strength, removal of both isoforms from the PSD by NMDA opens a window of opportunity for bi-directional synaptic modification. NMDA application to hippocampal neurons promotes recruitment of CYLD to the PSD and this effect is blocked by tatCN21 at a concentration that blocks CaMKII translocation. Moreover, CYLD and CaMKII co-immunoprecipitate from solubilized PSDs suggesting that CYLD could be recruited to the PSD through its association with CaMKII. CaMKII promoted phosphorylation of CYLD in isolated PSDs, as well as an increase in deubiquitinase activity specific for some types of polyubiquitins. Mass spectrometric analysis identified three residues that become phosphorylated under conditions that activate CaMKII, and both phosphorylation of the same three residues and CYLD activation occurs upon incubation of CYLD with purified CaMKII. CaMKII-mediated recruitment and activation of CYLD at the PSD would thus result in selective removal of particular polyubiquitins from PSDsspecifically those that tag proteins for lysosomal degradation. Thus, the net effect of CaMKII-mediated up-regulation of CYLD at the PSD would be to re-direct proteins away from the lysosomal. Our results reveal a novel downstream effect of CaMKII at the synapse and point to CYLD as a potential target for pharmacological intervention.